1. Field of Disclosure
The present disclosure relates to assay analysis, and, more specifically, to methods and systems for analyzing assay results using enhanced dynamic ranges.
2. Description of Related Art
Antibody-based immunoassays are used for applications such as biomarker verification or validation. One modern medical diagnostic testing technique is an enzyme-linked immunosorbent assay (ELISA). Generally, in an ELISA, a capture antibody is printed in the bottom of a reaction well in an assay substrate. An assay substrate is a surface upon which various chemical and/or biological analyses can be performed. Examples of an assay substrate include microarray plates, glass slides, and microtiter plates. A microtiter plate is a flat plate that has multiple “wells” formed in its surface. Each reaction well can be used as a small test tube into which various materials can be placed to perform biochemical analyses.
In an ELISA, the capture antibody has specificity for a particular antigen for which the assay is being performed. A sample to be analyzed is added to the well containing the capture antibody, and the capture antibody “captures” or immobilizes the antigen contained in the sample. A detect antibody is then added to the well, which also binds and/or forms a complex with the antigen. Further materials are then added to the well that cause a detectable signal to be produced by the detect antibody. For example, when light of a specific wavelength is shone upon the well, the antigen/antibody complexes will fluoresce. The amount of antigen in the sample can be inferred based on the magnitude of the fluorescence. In another example, a compound can be added to the well that causes the detect antibody to emit light within a predetermined wavelength (e.g., 400-500 nm) when properly energized by a suitable source. This light can be read by an optical detector, such as a charged-coupled device (CCD) camera or CMOS sensors, to measure the optical brightness or intensity of the emitted light. During an ELISA, the absorbency, fluorescence, or electrochemical signal of the well can be measured with suitable detection and processing equipment and compared with a standard to determine the presence and quantity of the sample antigen.
Assay performance involves the ability of the assay to precisely and accurately detect analytes in the sample. A singleplex immunoassay quantifies one analyte per assay, while a multiplex assay simultaneously measures multiple analytes in a single assay. Multiplex assays are used, for example, in functional genomics experiments that detect the presence of biomolecules of a given class (e.g., mRNAs, proteins) within a biological sample.
Factors that can influence the accuracy of assay detection include antibody pairs, binding between antibody and capture surface, signal amplification, and range of signal linearity detection. Because more variables are involved with multiplex assays as compared to singleplex assays, multiplex assays can be more prone to error. For example, cross-reactivity can occur in multiplex wells, as multiple pairs of capture antibodies and detectors are mixed in a single reaction well. Both pre-analytical error, for example, sample degradation or matrix heterogeneity, and analytical error can affect the accuracy and reliability of the detection signal.